Enriched platelet wound healant

ABSTRACT

An improved platelet gel wound healant, and methods of preparation and use thereof for healing wounds are disclosed. The improved wound healant comprises a therapeutically effective amount of activated growth factors and ascorbic acid with optional one or more additional anti-oxidant such as vitamin A and/or E, and optional one or more antibiotics.

CROSS-REFERENCE

[0001] This application is a continuation-in-part of U.S. applicationSer. No 09/424,523, filed Nov. 23, 1999, which is a continuation-in-partapplication of International application No. PCT/US99/02981, filed Feb.13, 1999, which claims priority from U.S. provisional application Ser.No. 60/090,167 filed Jun. 22, 1998, and U.S. provisional applicationSer. No. 60/097,897 filed Aug. 26, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates to improved platelet enrichedcompositions for wound treatment and methods of making and use thereof.

BACKGROUND OF THE INVENTION

[0003] There have been many different substances and methods developedin the past for treating wounds, depending upon the type and locationand severity of the wound. A wound is generally defined as an injury toan area of the body of a human or animal. Although injury to the surfaceof the skin is the most well known type of wound, the surfaces ofinternal organs may also be wounded, such as during surgery, rupture ofthe spleen or liver, or resulting from traumatic blows to the bodysurface in the vicinity of an internal organ.

[0004] Medical practice characterizes wounds as chronic or acute,according to the persistence and severity of the wound. A chronic woundis one that is prolonged or lingering, rather than promptly healed. Anacute wound is one that occurs relatively quickly, and heals relativelyquickly as well. Tissue wounds may have a wide spectrum ofmanifestations, as small as merely an abnormal microscopic tear orfissure in tissue (or a surface thereof), or as large as the abrasion orablation of the skin covering a substantial portion of the body, such asin a burn victim. Acute wounds covering a large or movable surface areusually the most difficult to guard from infection, and to heal.

[0005] Blood and bodily fluids include various substances that affectwound healing. The blood is the primary medium for delivering healingagents to the wound site, and for transporting foreign or harmfulsubstances away from the wound. Whole blood is primarily comprised ofthree main types of cells suspended in a protein rich solution known asplasma. The three main cell types in whole blood are erythrocytes(a.k.a. red blood cells), leukocytes (a.k.a. white blood cells) andthrombocytes (a.k.a. platelets). The red blood cells are theiron-containing cells that facilitate the transport and transfer ofoxygen to body tissue, and the removal of carbon dioxide. The whiteblood cells perform a variety of functions such as phagocytosis offoreign bodies and production of antibodies, and are primarilyresponsible for fighting infection and foreign substances within theblood or wound site. Platelets perform many functions such as pluggingleaks in blood vessels and helping begin the process leading to theformation of a blood clot; platelets contain substances known as growthfactors that facilitate the formation of new tissue.

[0006] Although there are several methods for separating whole bloodinto its various components, one of the most convenient and expeditiousmethods is accomplished by differentially centrifuging blood or some ofits components (i.e., apheresis). Using apheresis, the red and whiteblood cells and plasma may be separated out and returned to the donor'sor patient's body, leaving the sequestered platelets in essentiallyconcentrated form for use in wound healing techniques. From bloodextracted from a patient, the platelets may thus be obtained andactivated for use on the same patient; methods of using a patient's ownblood are called “autologous” or “autogenic” donor methods. Methodsusing blood donated by one or more third parties for use by a patientare called “homologous” or “heterologous” donor methods, or collectivelycalled “allogenic” methods.

[0007] One of the proteins suspended in plasma is fibrinogen, whichreacts with substances released into (or attracted by) wound sites toproduce sticky strands of fibrin. Such reactions result in the crosslinking of the fibrin strands to form a mesh that holds and supports thedeposit or growth of other tissue materials at the wound site.

[0008] The wound healing process is generally considered to occur inseveral stages, generally known as the healing cascade. After tissueinjury, platelets are among the first cells to appear in the vicinity ofthe wound. Activation of a platelet by an agonist such as thrombin, orother agonists such as those listed elsewhere herein, leads to therelease of granule material from within the platelet. Such granulationactivation results in the release of proteins known as growth factors,primarily concentrated in the alpha granules of platelets. Thesereleased growth factors stimulate the formation of new tissue; whenapplied to wounds, growth factors have been known to increase the rateof collagen laydown, vascular ingrowth, fibroblast proliferation andoverall healing. The release of a protein known as platelet-derivedgrowth factor (PDGF) is a chemotactic signal for monocytes, neutrophilsand fibroblasts which then move into the wound, to begin theinflammatory stage of the healing process. During this time, monocytessecrete a number of factors including PDGF and transforming growthfactor-β1 (also found in platelets), which recruits and activatesfibroblasts, to begin the repair stage of the healing process.Subsequently, wound healing continues through the process of collagenremodeling within the wound.

[0009] Based upon the foregoing general scientific principles, alreadyknown in the field are wound sealants made from biological materialsobtained primarily from tissue other than blood platelets. An example iswound sealants such as “fibrin glue”, which often are essentially amixture of co-coagulants (thrombin and calcium), concentrated fibrinogenand other coagulation proteins. In most applications, the primary rolesof fibrin glue are to seal wound surfaces to prevent loss of blood andother body fluids after surgery, and to provide adhesion betweenadjacent tissue surfaces. These products form a hard, cast-like coveringover the area to be sealed, and tend to be non-yielding to limbmovement.

[0010] The production of fibrin glue often requires obtaining fibrinogenfrom blood through a process known as cryoprecipitation, including bothfreeze-thaw cycles and relatively lengthy centrifugation of plasma incontrolled environments, to concentrate the fibrinogen in sufficientlyfor use; the precipitant thus obtained is frozen to −20° to −30°centigrade before storage. These requirements make such materialsunsuitable for application during the course of surgery, especiallyemergency surgery without an hour or more lead time. Moreover, to theextent this process depends upon the use of autologous biologicalmaterials, using this process shortly before or during surgery mayresult in the loss of crucial bodily fluids during a time when thepatient's body is badly in need of such fluids. By contrast,substantially larger amounts of concentrated platelets can be moreconveniently obtained within a matter of minutes from more recentmethods of differential blood centrifugation not requiring freezing andwithout significant loss of bodily fluids.

[0011] While there has been much research concerning fibrin glue, thismaterial belongs to a separate field from the present invention,primarily because fibrin glues typically contain cryoprecipitatedproteins without platelets. The use of fibrin glue is discussedextensively in the scientific literature; for example, see thereferences cited in U.S. Pat. No. 5,585,007 issued to Antanavich et alon Dec. 17, 1996.

[0012] Wound treatment compositions derived from platelet enrichedconcentrates are known and possess certain advantages over materialswithout platelets such as fibrin glue. One reason is that natural woundhealing agents are released by the platelets. Further, the concentrationof platelets likewise allows for a concentrated amount of wound healingfactors. Additionally, to the extent that the wound healing compositionis made from the biological materials of the patient, the risksassociated with heterologous donors (such as disease, immunologicreactions, or the like) are eliminated. Representative examples ofplatelet derived wound treatment compositions are described for instancein Hood U.S. Pat. No. 5,733,545; Knighton U.S. Pat. No. 5,165,938; andGordiner U.S. Pat. No. 5,599,558.

[0013] Platelet concentrates are typically isolated by the process ofdifferential centrifugation which essentially allows separating thepatient's own blood into at least three different components: packederythrocytes (red blood cells), plasma and platelet concentrate.Platelet concentrate can be combined with a solution of either sodium orcalcium mixed with thrombin (“calcified thrombin”), whichinstantaneously form a composition of activated platelets that, whenmade with the necessary viscosity, can be utilized as a wound sealant.The chemical reactions and cascades that normally occur when thrombin isadded to the concentrated platelets are indeed complex. See, forinstance, Reeder, et al, in Proceedings of the American Academy ofCardiovascular Perfusion, Vol. 14, January 1993. Such wound sealantstypically set up into a hard mass covering the application site, therebysealing the site against further blood loss and external contaminants.

[0014] There are a number of disadvantages associated with conventionalwound compositions derived from platelet concentrates. For instance,activation of platelets leads to instantaneous hardening of the materialand thus requires the physician to both activate and apply the plateletcomposition to the wound site within seconds of activation. Also,certain platelet compositions must be applied to the wound site on adaily basis and thus require regular blood withdrawal from the patient.The presence of a hardened mass at the wound site is undesirable becauseit impedes oxygen transport into the wound which is necessary for tissuerepair. It may also create a favorable environment for the growth ofpathogenic anerobic bacteria.

[0015] Accordingly, an improved platelet enriched wound treatmentcomposition which avoids or diminishes the problems associated withtypical platelet enriched wound compositions would be desirable.

SUMMARY OF THE INVENTION

[0016] The present invention relates to an improved enriched plateletcomposition for wound treatment, a method of making and use thereof. Thecomposition comprises platelets and an effective amount of ananti-oxidant, preferably ascorbic acid (vitamin C) so as to delay orprolong the activated gelation period to allow the practitionersufficient time to apply the composition in liquid form to the woundsite and to prevent the material from forming a hard seal. Optionalantibiotics may be included in the improved composition to preventinfections at the wound site. The presence of the anti-oxidant,including vitamins and non-vitamin anti-oxidants, and other healingpromotion materials that do not detract from, substantially interferewith, or even destroy the different thrombin activation reactions. Theinventive platelet gels containing ascorbic acid retain a stable softgel-like consistency during and after topical application at the woundsite and avoid the requirement for daily reapplication.

[0017] While the inventive composition is preferably used for topicalapplication to the exterior surface of the chronic wounds such as ulcersof the feet of diabetics, the composition may be applied to facilitatethe healing of other wounds such as acute wounds such as burns. However,the composition of matter and the methods described herein are notlimited solely to topical application.

[0018] The inventive composition increases the amount of growth factorsin the wound, and thereby facilitates the promotion of the healing rate.This may be especially important in “wounded” patients, especially thosewith chronic wounds who may lack sufficient circulation to facilitatethe healing cascade. The invention described herein also facilitates thecovering of the wound area with a substance that prevents or helps toreduce infection caused by most bacteria; and to the extent that thewound treatment material is made from autologous blood or similarbiological materials, the invention described herein reduces the risksassociated with the use of the treatment materials made from biologicalmaterials obtained from one or more third parties. An autologous productavoids some of the common problems associated with the use of biologicalmaterials from third parties, such as (for example) screening to assurethat the donor was biologically or immunologically compatible with thepatient, and otherwise free of hepatitis, HIV and the like.

[0019] In most general terms, the invention described herein expands theuses for concentrated platelet materials, especially those in gel form,by improving the speed and convenience of making the composition; theinvention described herein also improves the performance of theconcentrated platelet composition, by making it more useable forapplications over longer periods of time, and by enhancing the woundhealing and infection fighting properties. For autologous platelet gelto be more useful, the gelatinous state must be capable of remainingstable for a reasonable period of time. One aspect of the presentinvention is to add an anti-oxidant to the platelet gel, such asascorbic acid.

[0020] Another aspect of the present invention involves adding one ormore antibiotic substance at one or more times during the processingperiod so that the resulting concentrated platelet composition containseither one or a variety of the antibiotics. The use of an antibiotic inconcentrated platelet compositions that enhances the complex healingcascade is indeed novel. The invention disclosed herein involves addingsuch substances in a manner that does not detract from, substantiallyinterfere with, or even destroy these different reactions, pH balancesand potency.

[0021] Another aspect of the present invention involves adding one ormore vitamins, in addition to ascorbic acid (vitamin C) to theconcentrated platelet gel. Vitamins are known to have wound healing andanti-oxidant properties. Representative examples of suitable, but nonelimiting, vitamins include vitamin E, vitamin A and other retinoids.

[0022] In yet another aspect of the invention, non-vitamin anti-oxidantsmay be included in the concentrated platelet gel. Non-limitingrepresentative examples of such anti-oxidants include β-carotene.

[0023] The method of making the invention in gel form described hereinincludes admixing at least one of the described additives with theplasma-poor concentrated platelets, a sufficient time before theaddition of calcified thrombin (or other preferably-calcified agonist)to allow the desired dispersion of such additive(s) in such compositionbefore gelation prevents further dispersion.

DETAILED DESCRIPTION OF THE FIGURES

[0024]FIG. 1 is an illustration summarizing the levels of response forall 62 patients with various wound types following treatment with thegel coagulum of the invention as described in Example 4. The resultsshow that 46.8% of all patients had 100% closure after receiving, onaverage, 2.24 treatments over an averaged time duration of 8.74 weeks.

DETAILED DESCRIPTION OF THE INVENTION

[0025] Before the present invention is described in detail, it is to beunderstood that the invention is not limited to the particularconfigurations, process and materials expressly disclosed herein; theinvention includes those that are implicit or inherent in thedisclosures set forth herein, and all legal equivalents of an element(s)or limitation(s) thereof. As an example, the biological materialsspecified herein may originate from a patient to be treated, from asingle third party, or from a plurality of third parties; moreover, saidthird parties may be of the same species as the patient, or of anotherspecies, so long as the wound treatment material derived from suchbiological materials is biocompatible with the patient. As anotherexample, when the invention calls for a particular substance, it issufficient to use any form of that substance having thecharacteristic(s) needed to satisfy the stated need; for instance,unless the context indicates otherwise, a need for growth factors may besatisfied by providing isolated growth factors or those that areincluded in platelets or other types of cells, and/or combinationsthereof. Similarly, the process deployed to obtain the growth factorsmay be any process that satisfactorily does so, regardless of whether itincludes centrifugation.

[0026] It is also to be understood that the terminology used herein isnot intended to be limiting, since the scope of the present inventionwill be limited only by the claims and equivalents thereof. Also, asused herein, the singular forms include the plurals, and vice versa,unless the context indicates otherwise.

[0027] For the sake of simplicity and to give the claims of this patentapplication the broadest interpretation and construction possible, thefollowing definitions will apply:

[0028] (a) The phrase blood collecting or blood extraction (or similarphrase) includes techniques, materials and apparatus known in the field,such as (for example) inclusion of anticoagulation materials, the use ofblood drawing and infusion apparatus.

[0029] (b) The phrase growth factor means any material(s) promotinggrowth of a tissue.

[0030] (c) The term thrombin may include calcified thrombin, inparticular, about 5,000 units of thrombin per 5 ml of 10% of aqueouscalcium chloride solution; it may include calcified bovine thrombin aswell as autologous thrombin, allogeneic thrombin or recombinant humanthrombin.

[0031] (d) The term viscosity means those characteristics of thespecified material(s) determining the degree of gelation, such as (forexample) the firmness or hardness of the material, or the degree towhich the material resists flowing like a fluid.

[0032] (e) The term therapeutically effective amount means the amount oramounts of the constituent elements or combination thereof necessary toenhance wound healing such as, for example, the reduction in the volumeor surface area of a wound, the increase in the amount of granulationtissue or other biological material facilitating collagen laydown,vascular ingrowth, fibroblast proliferation or overall healing; all ofthe versions of the invention described herein are assumed to have thetherapeutically effect amount(s) of constituent substances, orcombinations thereof.

[0033] (f) The term anti-oxidant refers to any material(s) havinganti-oxidant properties. Anti-oxidant would include, without limitation,vitamins such as vitamins A and E and non-vitamins such as -carotene.

[0034] Also for the sake of simplicity, the conjunctive “and” may alsobe taken to include the disjunctive “or”, and vice versa, whenevernecessary to give the claims of this patent application the broadestinterpretation and construction possible. Likewise, when the plural formis used it may be taken to include the singular form and vice versa.

[0035] In most general terms, the invention includes a wound healantcomposition comprising activated growth factors and ascorbic acid. Inthe prevalent version of the invention, said growth factors are includedwithin platelets. The body produces many substances generally known asgrowth factors, and these growth factors are contemplated for use in thepresent invention. The preferred growth factors for use in the presentinvention are selected from the group consisting of platelet-derivedgrowth factor (PDGF), platelet-derived angiogenesis factor (PDAF),vascular endotheial growth factor (VEGF), platelet-derived epidermalgrowth factor (PDEGF), platelet factor 4 (PF-4), transforming growthfactor β (TGF-B), acidic fibroblast growth factor (FGF-A), basicfibroblast growth factor (FGF-B), transforming growth factor α (TGF-A),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), βthromboglobulin-related proteins (BTG), thrombospondin (TSP),fibronectin, von Wallinbrand's factor (vWF), fibropeptide A, fibrinogen,albumin, plasminogen activator inhibitor 1 (PAI-1), osteonectin,regulated upon activation normal T cell expressed and presumablysecreted (RANTES), gro-α, vitronectin, fibrin D-dimer, factor V,antithrombin III, immunoglobulin-G (IgG), immunoglobulin-M (IgM),immunoglobulin-A (IgA), a2-macroglobulin, angiogenin, Fg-D, elastase,keratinocyte growth factor (KGF), epidermal growth factor (EGF),fibroblast growth factor (FGF), tumor necrosis factor (TNF), fibroblastgrowth factor (FGF) and interleukin-1 (IL-1), Keratinocyte GrowthFactor-2 (KGF-2). and combinations thereof. One of the importantcharacteristics common to each substance, supporting the inclusion ofeach in this particular group, is that each such substance is known orbelieved to enhance cell or tissue growth. Moreover, said substances, orvarious combinations thereof, are known or believed to function togetherin an unexpected synergistic manner to promote wound healing. Suitable,non-limiting, anti-oxidants useful in the invention include but are notlimited to vitamins such as vitamin C (ascorbic acid), vitamin E,vitamin A and other retinoids; and the carotenes such as β-carotene. Inpracticing this invention, ascorbic acid as anti-oxidant is particularlypreferred.

[0036] The presence of ascorbic acid in the concentrated platelet gelprovides a number of surprising and beneficial effects. Ascorbic aciddelays or prolongs the activated platelet gelation period to allow thepractitioner sufficient time to readily apply the gel compositiontopically to the wound site and to prevent the applied material fromforming an undesirable hard seal. The general amount of ascorbic acidthat may be used in preparing the inventive composition is sufficient toenhance the preservation of the soft gelatinous state of the final woundhealing composition. The amount of ascorbic acid generally rangesbetween about 10 mgs and about 100 mgs, preferably ranging between about20 mgs and about 65 mgs, per ml of platelet concentrate. Ascorbic acidis available as Cenolate® ascorbic acid injection USP (500 mg/ml, pH5.5-7.0; Abbott Laboratories, North Chicago, Ill., USA). This is asolution of sodium ascorbate that is prepared from ascorbic acid withsodium bicarbonate in water for injection.

[0037] The platelets are separated from the red blood cells and whiteblood cells of whole blood, primarily through differentialcentrifugation, although any suitable method for separating plateletsfrom whole blood may be employed in practicing this invention. Theoverall composition of the invention disclosed herein may containincidental amounts of white blood cells, due to the fact that theplatelets are rarely totally isolated from the other blood components.It is believed that the present invention contains only minimal or traceamounts of white blood cells; it is believed that the white blood cellcount of the present invention typically will be below about 3 times 10⁷cell/ml. The bioactive material in the invention is almost exclusivelyfrom platelets. The range of the mean platelet volume of the plateletsbeing sequestered is in the range of about 6.6 to 8.4 femtoliters, withan average of about 7.7 femtoliters; this may indicate that theplatelets being sequestered are relatively larger or younger than theoverall population of platelets.

[0038] Activation of growth factors may occur in a variety of manners,by a variety of substances known as activators or agonists. In theinvention described herein, said activation results from the inclusionof an activator or agonist selected from the group consisting ofthrombin, collagen, serotonin, adenosine diphosphate (ADP) andacetylcholine (ACH), and combinations thereof. In a particular andpreferred version of the invention, said growth factors are includedwithin concentrated platelets, and said activation results from theinclusion of thrombin. One of the important characteristics common toeach substance, supporting the inclusion of each in this particulargroup, is that each such substance is known or believed to enhance cellor tissue growth in addition to the ability to activate platelets.Moreover, said substances, or various combinations thereof, are known orbelieved to function together in an unexpected synergistic manner topromote wound healing.

[0039] The activator or agonist added to the platelet concentrate is inan amount sufficient to facilitate the formation of the coagulum (gel)having a predetermined viscosity while sufficiently activating growthfactors present in the composition. In the preferred case where thrombinis employed as the activator to produce a final soft gel woundcomposition in a soft gel form, the amount of thrombin generally rangesbetween about 100 U and about 10,000 U, preferably about 900 U and about1100 U, most preferably about 1000 U per 8 cc of platelet concentrate.Thrombin is available as Thrombogen® thrombin, topical USP (bovineorigin) in vials containing 5000 units thrombin (Johnson & JohnsonMedical Inc., Arlington, Tex., USA).

[0040] Thrombin activation requires the presence of certain cofactorssuch as sodium or calcium. In practicing this invention, thrombinactivation preferably occurs in the presence of calcium ions. Calciumions are generally added to the platelet concentrate as a salt solutionto provide a final concentration generally ranging between about 0.1mg/ml and about 10 mg/ml, preferably 1 mg/8 cc of platelet concentrate.Suitable calcium salts include, without limitation, CaCO₃, and CaSO₄. Apreferred calcium salt for use in the invention is CaCl₂. CaCl₂ isavailable as calcium chloride injection, USP 10% (American RegentLaboratories, Inc., Shirley, N.Y., USA).

[0041] The invention is not limited to autologous biological materials,such as collection of concentrated platelets from the wounded's ownbiological material. The invention encompasses the use of biologicalmaterials obtained from one or more third parties, that need not be ofthe same species as the patient whose wound is being treated with thewound healant composition described herein unless bioincompatibilitywould result from the use of such third party biological materials.

[0042] In one general version of the invention, the wound healantcomposition includes concentrated platelets, thrombin and ascorbic acid.Ascorbic acid is known to have preservative properties, unless it isbroken down such as occurs after exposure to sunlight or another sourceof ultraviolet (UV) light rays. However, most versions of the inventiondescribed herein are covered by bandages or otherwise shielded from UVrays almost immediately after application to the wound site.

[0043] Since the admixture of thrombin or other agonists will activategrowth factors, the thrombin (or other agonists/activators) shouldusually be the last substance to be mixed immediately before it isdesired that the gelatinous state be set up.

[0044] Another version of the invention includes the inclusion of atleast one other antioxidant vitamin, such as vitamin E, vitamin A andother retinoids in the admixture, in addition to or in substitution ofthe ascorbic acid. Although the wound healant composition could includea combination of one or more vitamins, one version of the inventionmerely includes vitamin A in addition to or in substitution of theascorbic acid. Vitamin A has been known to counteract a side effect ofsome treatments using steroids, namely, the depressed reactivity of thebody immune system to stimuli. Furthermore, vitamin A is known orbelieved to inhibit or decrease the bioactivity of manganese, magnesiumand copper in the cellular and interstitial environment; said elementsare known or believed to be active or instrumental in the laying down ofkeloids and scar tissue. (See, “Effects of Pantothenic Acid and AscorbicAcid Supplementation of Human Skin Wound Healing Process” by Vaxman etal., Eur. Surg. Res. 1995, 28:4, 158-166.) The versions of the inventiondescribed herein containing vitamin A accordingly are believed topromote healing without as much scarring or keloid formation. In anotherversion, vitamin E, known to facilitate healing, may be used. In anyevent, the vitamins disclosed herein (or combination thereof) appear toenhance wound healing in an unexpectedly synergistic manner in additionto their roles in gel formation. The general amount of vitamin that maybe used in preparing the inventive composition is one that is sufficientto enhance the preservation of the soft gelatinous state of the finalwound healing composition.

[0045] Since many wound sites are either already infected with bacteriaor are susceptible to such infection, it is desirable that a woundhealant composition be capable of either killing bacteria or preventingthe mobility or reproduction of bacteria. The invention described hereinincludes a wound healant composition comprising concentrated platelets,thrombin and at least one antibiotic. In particular, the inventionincludes a wound healant wherein said antibiotic is bacteriocidal to atleast the Pseudomonas and Klebsella genera of bacteria, which areprevalent at wound sites and difficult to guard against. Alternatively,said antibiotic is selected from the group consisting of a neosporin,vancomycin and gentamycin, and combinations thereof. One of theimportant characteristics common to each substance, supporting theinclusion of each in this particular group, is that each such substanceis known to kill said bacteria.

[0046] As indicated above, the invention may include a wound healantcomposition comprising concentrated platelets, thrombin, ascorbic acid,at least one retinoid and at least one antibiotic bacteriocidal to thePseudomonas and Klebsella genera of bacteria.

[0047] Aside from the wound healant substance to be applied to woundsites, the invention further includes a method of making a healantcomposition which comprises providing a platelet enriched solutionincluding an effective amount of anti-oxidant such as ascorbic acid andan activator effective amount of a cofactor, e.g., a divalent cationcofactor; and activating the solution with an activator or agonist asdescribed above.

[0048] Regardless of the amount of set up gelation time, the presentinvention includes an anti-oxidant that allows the gel to retain itsviscosity for a longer duration. For example, ascorbic acid is believedto preserve the longevity of the gel viscosity. Another method of makingthe wound healing composition includes the steps of mixing activatedgrowth factors with ascorbic acid. Said activated growth factors may beobtained in a variety of ways, such as by the steps of sequesteringconcentrated platelets from blood and mixing thrombin with saidplatelets. The ascorbic acid should be in sufficient amount to enhancethe preservation of the gelatinous state of the final wound healingcomposition, and said thrombin should be in sufficient amount tofacilitate formation of the coagulum (gel) having the desired level ofviscosity while sufficiently activating growth factors present in thecomposition and the wound.

[0049] In one preferred version of the composition, ascorbic acid (forinstance, a 1 ml of a Cenolate® ascorbic acid solution (500 mg/ml;Abbott Laboratories, Inc., North Chicago, Ill., USA) is admixed withabout 5 cc and 10 cc, preferably about 8 cc of concentrated platelets,then about 500 U and about 10,000 U, preferably about 1000 U ofcalcified thrombin (prepared by the addition of 1 cc of 10% CaCl₂/1000 Uof thrombin solution] is admixed into the platelet/ascorbic acidadmixture. However, other ratios of concentrated platelets:ascorbicacid:thrombin may be useful, depending upon the desired amount ofhealing agents, gelation time, gel viscosity and longevity. The methodof making a wound healant may further include, prior to orcontemporaneous with mixing said thrombin, mixing at least one of theaforementioned vitamins in sufficient amount(s) to further enhance woundhealing. Alternatively, said method may include, prior to orcontemporaneous with mixing said thrombin, mixing at least one of theaforementioned antibiotics in sufficient amount(s) to reduce infectionby bacteria.

[0050] Besides a method of making a wound healant composition, theinvention described herein may also include a method of treating awound, comprising the steps of applying a sufficient amount of acomposition of matter comprising growth factors and ascorbic acid toenhance healing of the wound. Said method of treating a wound mayinclude the use of any of the compositions described herein; it may alsoinclude the use of any composition made by any of the methods describedherein.

[0051] Once applied to a wound, the composition may remain on the woundfor as long as 5 days, and perhaps longer depending upon thecircumstances such as the location of the wound and other woundcharacteristics. Although the composition and method described hereinare especially useful for the treatment of chronic wounds, they may alsobe useful in the treatment of acute wounds.

EXAMPLE 1 Preparation of Platelet Enriched Wound Healant

[0052] (a) Whole Blood Method

[0053] One manner of making the plasma-poor concentrated platelets ofthe present invention is to collect about 450 ml of whole blood inanticoagulant (such as sodium citrate or any similar anticoagulant knownin the field). That blood is then centrifuged in a MCS® Plus LN-9000centrifuge (Haemonetics, Inc.) using an LN 994 disposable collection kit(Haemonetics, Inc.) at about 2,400 rpms for a duration of about 20minutes to separate out a band of: (a) plasma and most white bloodcells; (b) platelets (and incidental white blood cells); and (c) redblood cells. The platelet portion is then re-centrifuged to furtherseparate out plasma (and incidental white blood cells) at 4,800 rpms fora duration of about 7½minutes.

[0054] The final yield is about 45 ml of plasma-poor concentratedplatelets (in trace or incidental amounts of residual plasma and whiteblood cells). Both the plasma/leukocyte portion and the red blood cellportion may be reinfused back into the patient. The concentration of theplatelets fell within an acceptable platelet concentration rangingbetween about 350,000 and about 2×10⁶ cells/ml of platelet concentrate.

[0055] The plasma-poor concentrated platelets (8 ml) is then admixedwith 1 ml of aqueous ascorbic acid solution (concentration 500 mg/ml;Cenolate® ascorbic acid injection, USP, available from AbbottLaboratories, North Chicago, Ill., USA) in a sterile vial. The plateletmixture is then activated by the addition of 1 ml of a 10% CaCl₂/1000 Uthrombin (prepared by admixing 5 ml of aqueous 10% CaCl₂ (Calciumchloride injection, USP 10%, American Regent Laboratories, Inc.,Shirley, N.Y., USA) with 5000 U Thrombin (Thrombogen® Thrombin, topicalUSP (bovine origin)), 5000 U, Johnson & Johnson Medical Inc., Arlington,Tex., USA).

[0056] Depending upon the relative concentrations of the ingredients,the resulting mixture may be either a liquid, or it may set up as a hardmaterial or (preferably) as a gel having a viscosity dependant upon therelative amounts of thrombin and platelets; the relative concentrationsof calcified thrombin to platelets determines how quickly thecomposition sets up, and how hard it will eventually be. Some mixtureswill yield a composition that will set up in a gel in several seconds,whereas some mixtures will yield a composition that takes severalminutes to set up in a gel.

[0057] (b) Platelet Method

[0058] This example illustrates another method of making the compositionallows the extraction of blood, sequestering of plasma-poor concentratedplatelets, mixing of additives and return of unused blood components tothe patient, all in about 20 to 30 minutes and by making only onepuncture in the patient. Approximately 125 to 250 ml of blood isextracted from a patient, with the blood drawing apparatus optionallyremaining in place connected to the patient (for later use in returningunused blood components to the patient). That blood is transferred to aLathum bowl and, using the same centrifuge described in above method(a), centrifuged at about 4,800 rpms until a band (or similar grouping)of plasma forms at the upper periphery (about 5 to 15 minutes), and aband (or similar grouping) of red blood cells forms at the bottom of thebowl; the center is comprised of plasma-poor concentrated platelets. Theplasma band is removed for return to the patient, and the remainingblood components are again centrifuged at that speed (and sufficientduration), further removing plasma and white blood cells from theplasma-poor concentrated platelets. The plasma-poor concentratedplatelets are then removed for mixing with the other additives describedherein (thrombin, ascorbic acid and/or retinoids). See method (a) above.

EXAMPLE 2 Case Study

[0059] Case Study

[0060] Patient P is a 57 year old white male truck driver with a rightheel diabetic ulcer of 11 month's duration. His treatment regimen hasconsisted of rest, off-loading and daily wound cleansing with soap andwater followed by application of gauze dressing. Carrasyn gel wasordered for a brief period, without improvement. Upon referral to anoutpatient physical therapy department for wound treatment, P's currenttherapy consists of weekly sharp debridement, wet saline gauze and totalcontact cast. P has history of hypertension, which is controlled atpresent time. He has a 15-year history of diabetes mellitus withneuropathy, which is controlled with oral hypoglycemic elements.

[0061] P began treatment with the invention disclosed herein. After hiswound was sharply debrided, a gel coagulum prepared as described inExample 1 was applied and the wound was covered with a wet salinedressing. A total contact cast was then applied and left intact for oneweek. At the conclusion of week 1, the cast was removed, the wound sitecleansed and recovered with wet dressing; the limb was re-cast for week2. After the conclusion of week 2, the same procedure was followed,except that the gel coagulum was again applied to the wound site beforecovering with wet dressing.

[0062] After the conclusion of week 3 the same procedure as for week 2was followed. This regimen continued for a total of 36 days. Table 1below contains the data reflecting the reduction in wound site volumeand surface area during weeks 1 through 4. TABLE 1 Week # Volume (mm³)Area (mm²) 0 3121 674 1 1561 562 2  279 301 3  26 282 4  15 159

EXAMPLE 3 Case Study

[0063] In this Example, five patients that entered into this study werereferrals by their physicians. The patients were then screened using theexclusion, inclusion criteria. The patients selected for study had anulcer of the lower extremity that had not healed after four to sixmonths of treatment either with traditional wound care alone, or withtraditional care plus Regranex¹.

[0064] All five of the study patients had a platelet count of 100,000cells/mm³ or greater, and had a hemoglobin >10 g and a HCT of 30% orgreater. The patients were evaluated for infection in the wound, and forosteomyelitis. None of the patient studied showed signs of infection, orbone involvement.

[0065] Aggressive debridement to essentially change the chronic wound toan acute one was used. The ulcer and surrounding callus were completelyexcised down to normal uninvolved tissue. All subjects were treated asoutpatients. All patients agreed to be totally non-weight-bearing. Withthe exception of one, patients were supplied with a half-shoe thattransferred weight to the unaffected area of the foot. The one patientnot fitted with the half-shoe was fitted with a full case of the lowerleg. Direct questioning of patients and family assessed the complianceissue. Only one patient proved to be non-compliant. Patient 4's bloodsugar exceeded 400 mg/dl and she became disoriented and walked on herfoot as post treatment day 2. This resulted in a re-treatment of patient4 ². Table 2 shows the wound size and volume at the commencement andconclusion of treatment with the gel coagulum disclosed herein (Example1). Closure, on the average, took less than four weeks. The averagewound was brought to 99% closure in 25 days. TABLE 2 Volume (mm³) Area(mm²) Pt # Start End Start End 1 3121 mm³ 15 mm³ 674 mm² 159 mm² 2 358mm³ 0.0 mm³ 65 mm² 4 mm² 3 293 mm³ 3.0 mm³ 63 mm² 3 mm² 4 192 mm³ 18 mm³104 mm² 39 mm² 5 336 mm³ 0.0 mm³ 181 mm² 0.0 mm²

EXAMPLE 4 Expanded Clinical Case Study

[0066] In this Example, the gel composition prepared in accordance withExample 1 was evaluated in 62 patients with a variety of chronic woundtypes resulting from several causes, including diabetic complications,trauma including wounds resulting from surgical or medical procedures,pressure (decubitus sores), and spider bites. The patients were dividedinto four (4) clinical groups, and were all categorized according towound duration, wound type, date of treatment initiation and startingwound volume, extent of wound closure and time duration to reach woundclosure. The results are tabulated in Table 3 below and are summarizedgraphically in FIG. 1.

[0067]FIG. 1 is an illustration summarizing the levels of response forall 62 patients with various wound types following treatment with thegel coagulum of the invention. The results show that 46.8% of allpatients had 100% closure after receiving, on average, 2.24 treatmentsover an averaged time duration of 8.74 weeks. The breakdown of theresults according to wound types is shown in Table 5 below. TABLE 3 No.Successful patients^(a)/No. Wound Type total patients % Woundclosure^(b) Diabetic 25/29 86 Venous  7/15 47 Pressure 3/5 60 Trauma 5/863 Spider 2/2 100 

[0068] BENNETT WOUND THERAPY Initial Current # of Week's to Healing IDWound Duration Wound Type 1^(st) TX Date Wound Vol. Wound Vol. TX's %Closure Date or Current Closure Mcla 12 months Diabetic 8/2/1999 853 mm³0 2 100% 8/30/1999 4 Rcoo 1 month Venous 9/8/1999 2660 mm³ 0 1 100%10/8/1999 4.5 Sdlc 12 months Diabetic 10/1/1999 586 mm³ 0 2 100%######## 6.5 Mdrl 48 months Diabetic 2/22/2000 22811 mm³ 0 1 100%4/25/2000 9 Near NA Surgical 12/20/1999 583625 mm³ 0 1 100% 3/6/2000 11Rfau 1 month Venous 10/20/1999 600 mm³ 0 2 100% ######## 8 Dgra 2 monthsDiabetic 9/8/1999 160 mm³ 0 3 100% 1/11/2000 18 Jhor 1 month Diabetic10/19/1999 286 mm³ 0 4 100% 1/11/2000 12 Ajoh 288 mo. (24 yrs) Venous8/9/1999 1600 mm³ 0 4 100% ######## 10 Djon <1 month Burn 9/22/1999 2761mm³ 0 1 100% ######## 3 Plal 1 month Diabetic 10/8/1999 264 mm³ 0 1 100%######## 7.5 Amcc 2 months Diabetic 1/13/2000 1700 mm³ 0 1 100% 4/4/200011.5 Dvil <1 month Diabetic 1/17/2000 933 mm³ 0 3 100% 3/29/2000 10 Pwhl1 month Diabetic 10/13/1999 1000 mm³ 0 1 100% ######## 5 Cwho 2 monthsSpider 7/7/1999 18676 mm³ 0 2 100% ######## 14 Myat 1 month Diabetic9/23/1999 595 mm³ 0 3 100% 1/24/2000 17 Jbea 180 mo. (15 yrs) Diabetic3/14/2000 440 mm³ 102 mm³ 7  77% 6/21/2000 14 Dbru 1.5 months Surgical5/2/2000 164749 mm³ 56028 mm³ 1  66% 5/16/2000 2 Wbur NA Diabetic3/29/2000 32099 mm³ 580 mm³ 6  98% 6/8/2000 10 Mbye 4 months Diabetic1/11/2000 3001 mm³ 0 5 100% 5/16/2000 18 Ccan 2 months Pressure4/20/2000 800 mm³ 0 4 100% 6/22/2000 9 Bcla 1 month Spider 4/25/200014007 mm³ 0 2 100% 6/20/2000 8.5 Ccle 3 months Decubitus 2/24/2000 1900mm³ 3169 mm³ 7  0 6/8/2000 15 Wcot 18 months Venous 4/25/2000 2701 mm³583 mm³ 3  78% 6/1/2000 5 Ffer 8 months Diabetic 2/7/2000 4168 mm³ 160mm³ 6  96% 6/22/2000 19.5 Dshe 10 months Diabetic 4/20/2000 12272 mm³1248 mm³ 4  90% 6/22/2000 9 Jshe 3 months Diabetic 5/16/2000 144 mm³ 17mm³ 2  88% 6/6/2000 3 Wsme 14 months Diabetic 8/4/1999 2667 mm³ 5 mm³ 2 99% 6/13/2000 45 Lsmi 8 months Diabetic 2/15/2000 128 mm³ 0 7 100%6/20/2000 18 Wspu 144 mo. (12 yrs) Venous 6/30/1999 18009 mm³ 666 mm³22   96% 6/21/2000 51 Fbla 4 months Diabetic 5/25/2000 306 mm³ 133 mm³ 2 57% 6/22/2000 4 Rwil 9 months Venous 2/24/2000 986 mm³ 28 mm³ 8  97%6/13/2000 16 Jwoo 4 months Club Foot 5/30/2000 120 mm³ 10 mm³ 1  92%6/20/2000 3 CALDWELL Cwin 3 months Pressure 3/16/2000 273836 mm³ 16008mm³ 7  94% 6/6/2000 11.5 Jspe 12 months Venous 6/8/2000 28814 mm³ 7803mm³ 1  73% 6/13/2000 1 Jemm 6 months Diabetic 3/30/2000 933 mm³ 27 mm³ 3 97% 5/2/2000 5 Jker NA Pressure 6/8/2000 2251 mm³ 1067 mm³ 1  53%6/13/2000 1 Rhow 4 months Pressure 4/6/2000 12006 mm³ 0 2 100% 6/14/200010 Mjon 12 months Diabetic 5/3/2000 1600 mm³ 373 mm³ 3  77% 6/15/2000 6HSM-METH Wenn 105 mo. (9 yrs) Arterial 5/8/2000 14967 mm³ 14791 mm³ 10.01%  5/16/2000 1 Bbou 4 months Trauma 5/2/2000 13147 mm³ 520 mm³ 4 96% 6/19/2000 7 Bbou 4 months Trauma 5/2/2000 598 mm³ 0 mm³ 3 100%6/1/2000 4 Gger Est. 20 years Venous 4/27/2000 8590 mm³ 1920 mm³ 2  78%6/12/200  7 Gger Est. 20 years Venous 4/27/2000 793 mm³ 659 mm³ 2  17%6/12/2000 7 Gger Est. 20 years Venous 4/27/2000 815 mm³ 1073 mm³ 2  0%6/12/2000 7 Kcar 6 months Diabetic 4/26/2000 640 mm³ 47 mm³ 3  93%6/7/2000 6 Vols 6 months Trauma 4/27/2000 2534 mm³ .667 mm³ 2  99%6/26/2000 9 Mjos 193 mo. (16 yrs) Venous 4/26/2000 1664 mm³ 3241 mm³ 3 0% 6/12/2000 7 Baug NA Trauma 6/15/2000 1814 mm³ 400 mm³ 1  78%6/19/2000 4 days Alev NA Venous 5/23/2000 2977 mm³ 1323 mm³ 2  56%6/15/2000 3 Mmad 18 months Venous 5/4/2000 1000 mm³ 469 mm³ 3  53%6/8/2000 4 Cdic NA Trauma 4/27/2000 2871 mm³ 2401 mm³ 2  16% 6/8/2000 5LRCF Rall 36 mo. (3 yrs) Diabetic 11/13/1999 2941 mm³ 0 1 100% 12/8/19993 Catk 24 months Diabetic 3/1/2000 13340 mm³ 27 mm³ 2  99% 5/3/2000 8Rbak 8 months Diabetic 2/11/2000 133 mm³ 6 mm³ 1  95% 3/2/2000 3 Habr 4months Diabetic 7/6/1999 880 mm³ 0 1 100% 8/12/1999 5 Bbol 144 mo. (12yrs) Venous 12/22/1999 800 mm³ 0 3 100% 3/6/2000 10 Jbrl 6 monthsDiabetic 12/1/1999 32 mm³ 0 1 100% 12/6/1999 5 days Fbri 2 months Venous5/17/2000 3601 mm³ 200 mm³ 1  94% 6/21/2000 4 Shui 5 months Surgical4/18/2000 1467 mm³ 0 2 100% 6/7/2000 7 Rmar 24 months Diabetic 3/29/200021 mm³ 0 1 100% 4/25/2000 4 Rmcd 4 months Diabetic 12/31/1999 4 mm³ 0 1100% 2/7/2000 5

What is claimed:
 1. A wound healant composition comprising atherapeutically effective amount of activated platelet concentrate andascorbic acid.
 2. A wound healant described in claim 1 , wherein saidascorbic acid is present in an amount ranging between about 20 mg andabout 65 mg per ml of activated platelet concentrate.
 3. A wound healantdescribed in claim 1 , wherein said activated platelet concentrateresults from the inclusion of an agonist to activate a plateletconcentrate.
 4. A wound healant described in claim 3 , wherein saidagonist is selected from the group consisting of thrombin, collagen,serotonin, adenosine diphosphate (ADP) and acetylcholine (ACH), andcombinations thereof.
 5. A wound healant described in claim 1 , whereinsaid growth factors are included within concentrated platelets, and saidactivation results from the inclusion of thrombin.
 6. A wound healantdescribed in claim 5 , wherein said concentrated platelets areautologous concentrated platelets and having a white blood cell count ofbelow about 3 time 10⁷ cells/ml.
 7. A wound healant compositioncomprising a therapeutically effective amount of concentrated platelets,ascorbic acid and thrombin.
 8. A wound healant composition comprising atherapeutically effective amount of concentrated platelets, at least oneanti-oxidant and thrombin.
 9. A wound healant composition described inclaim 8 , wherein said anti-oxidant comprises a retinoid, vitamin E, orβ-carotene.
 10. A wound healant composition described in claim 9 ,wherein said retinoid is vitamin A.
 11. A wound healant compositioncomprising a therapeutically effective amount of concentrated platelets,at least one antibiotic and thrombin.
 12. A wound healant described inclaim 11 , wherein said antibiotic is bacteriocidal to at leastPseudomonas and Klebsella bacteria.
 13. A wound healant described inclaim 11 , wherein said antibiotic is selected from the group consistingof a neosporin, vancomycin and gentamycin, and combinations thereof. 14.A wound healant composition comprising concentrated platelets, ascorbicacid, at least one retinoid, at least one antibiotic bacteriocidal to atleast Pseudomonas and Klebsella and thrombin.
 15. A wound healantcomposition comprising concentrated platelets in an amount rangingbetween about 350,000 and about 2×10⁶ cells/ml, ascorbic acid in anamount ranging between about 20 mg and about 65 mg/ml, thrombin in anamount ranging between about 100 U and about 10,000 U, preferably about900 U and about 1100 U, most preferably about 1000 U per 8 cc ofplatelet concentrate, and calcium chloride in an amount ranging betweenabout about 0.1 mg/ml and about 10 mg/ml.
 16. A wound healantcomposition as described in claim 15 , further comprising vitamin A andvitamin E in effective anti-oxidative amounts.
 17. A method of making awound healant composition, comprising the steps of mixing, intherapeutically effective amount(s), activated platelet concentrate withascorbic acid.
 18. A method of making a wound healant described in claim17 , wherein said activated platelet concentrate are obtained by thesteps of sequestering concentrated platelets and mixing thrombin withsaid platelets.
 19. A method of making a wound healant described inclaim 17 , said method further comprising, prior to mixing saidthrombin, mixing at least one retinoid in sufficient amount(s) tofurther enhance wound healing.
 20. A method of making a wound healantdescribed in claim 22 , wherein said retinoid is vitamin A in sufficientamount to reduce any non-responsiveness of the wounded's immune systemto stimuli, and vitamin E in sufficient amount to further enhance woundhealing.
 21. A method of making a wound healant described in claim 23 ,wherein vitamin and vitamin E are admixed with concentrated plateletsand ascorbic acid, then admixed with thrombin with calcium chloride. 22.A method of making a wound healant described in claim 17 , said methodfurther comprising, prior to mixing said thrombin, mixing at least oneantibiotic in sufficient amount(s) to reduce infection by bacteria. 23.A method of making a wound healant described in claim 22 , wherein saidantibiotic is at least bacteriocidal to Pseudomonas and Klebsellabacteria.
 24. A method of making a wound healant described in claim 22 ,wherein said antibiotic is selected from the group consisting ofneosporin, vancomycin and gentamycin, and combinations thereof.
 25. Amethod of making a wound healant, comprising the steps of, prior tomixing thrombin, admixing, in therapeutically effective amount(s),concentrated platelets, ascorbic acid, at least one retinoid and atleast one antibiotic bacteriocidal to at least Pseudomonas and Klebsellabacteria.
 26. A method of making a wound healant composition, comprisingthe steps of: extracting blood from a patient, centrifuging said blooduntil the appearance of an essentially separate band of plasma, anessentially separate band of red blood cells, and an essentiallyintermediate grouping comprised of concentrated platelets therebetween;removing said plasma band; centrifuging said remaining blood componentsat said speed an sufficient duration; removing said concentratedplatelets; and mixing, in therapeutically effective amount(s), saidconcentrated platelets with thrombin and ascorbic acid.
 27. A woundhealant prepared in accordance with claims 17-24.
 28. A wound healantprepared in accordance with claim 25 .
 29. A wound healant prepared inaccordance with claim 26 .
 30. A method of treating a wound, comprisingthe steps of applying a sufficient amount of composition of mattercomprising a therapeutically effective amount of activated plateletconcentrate and ascorbic acid to enhance healing of the wound.
 31. Amethod of treating a wound described in claim 30 , wherein saidactivation results from the inclusion of an agonist.
 32. The method oftreating a wound described in claim 31 , wherein said agonist isselected from the group consisting of thrombin, collagen, serotonin, ADPand acetylcholine (ACH), and combinations thereof.
 33. A method oftreating a wound described in claim 30 , wherein said growth factors arederived from concentrated platelets, and said activation results fromthe inclusion of thrombin.
 34. A method of treating a wound described inclaim 30 , wherein said concentrated platelets are autologousconcentrated platelet.
 35. A method of treating a wound described inclaim 30 , said wound healant composition comprising concentratedplatelets, ascorbic acid and thrombin.
 36. A method of treating a wounddescribed in claim 30 , said wound healant composition comprisingconcentrated platelets and thrombin, and further comprising at least oneretinoid.
 37. A method of treating a wound described in claim 36 ,wherein said retinoid is vitamin A.
 38. A method of treating a wounddescribed in claim 36 , wherein said retinoid is vitamin E.
 39. A methodof treating a wound described in claim 30 , said wound healantcomposition comprising concentrated platelets and thrombin, and furthercomprising at least one antibiotic.
 40. A method of treating a wounddescribed in claim 39 , wherein said antibiotic is bacteriocidal to atleast Pseudomonas and Klebsella bacteria.
 41. A method of treating awound described in claim 39 , wherein said antibiotic is selected fromthe group consisting of a neosporin, vancomycin and gentamycin, andcombinations thereof.
 42. A method of treating a wound described inclaim 30 , said wound healant composition comprising concentratedplatelets, ascorbic acid, at least one retinoid, at least one antibioticbacteriocidal to Pseudomonas and Klebsella bacteria, and thrombin.